NMDA receptors are Ca++-gating inotropic receptors. They are multi- subunit hetero-oligomers assembled from two classes (NR1 and NR2) of subunits. The one NR1 gene is processed into at least seven distinct splice variants and the four NR2 genes produce proteins that combine, in unknown combinations and stoichiometry, with NR1 to form receptors of distinct physiological characteristics depending on subunit composition. We have developed a battery of antibodies that can selectively recognize each of the subunits on immunoblots and can selectively immunoprecipitate a specific subunit. By solubilizing receptors in a gentle detergent, the subunit interactions remain intact and co- immunoprecipitation of multiple subunits with a specific antibody yields information regarding subunit associations in situ. This project proposes: (1) to determine the subunit combinations that assemble into NMDA receptors in various regions of the rat brain, (2) to generate cell lines stably expressing NMDA receptors of defined subunit composition that represent the major species of NMDA receptors expressed, (3) to examine the functional properties of NMDA receptors of defined subunit composition, (4) to measure the stoichiometry of NMDA receptor subunits in assembled NMDA receptors, and (5) to examine the possibility that agrin mediates tyrosine phosphorylation and/or aggregation of NMDA receptors. Results from these studies will increase our knowledge of the structure, function, location, and pharmacology of these receptors. Because NMDA receptors are involved in many important functions such as synaptic plasticity, which is vital for normal CNS development and may underlie the processes of learning, memory, and excitotoxicity, which may be involved in ischemic brain damage as well as several neurological diseases, drugs that affect the function of these receptors could be useful in the treatment of stroke,neurodegenerative diseases, and control of chronic pain to name a few examples. However, until we understand the various types and properties of the receptors that are expressed, side-effects will dominate any drugs that act via these receptors.